Identification of cellular sensors for key parameters such as lateral pressure in the lipid bilayer and degree of cytoplasm hydration will not only advance our basic understanding of cell physiology and mechanics, but can also be used in the development of bio-inspired sensor devices for probing the environment and for screening potential pharmaceuticals. Mechanosensitive Channel of Small Conductance (MscS) is a ubiquitous osmolyte release channel found in all phyla of organisms with cell walls. Esherichia coli MscS, the best understood representative, is directly activated by membrane tension and inhibited by increased crowding pressure of polymers in the cytoplasm. Crystal structures predict that the transmembrane domain of MscS senses tension, whereas the hollow cytoplasmic domain (cage) perceives crowding pressure and adjusts tension sensitivity and duration of opening according to the degree of cytoplasmic hydration. Additionally, due to the asymmetric position of the gate relative to the membrane midplane, MscS is more sensitive to lateral pressure/tension in the inner leaflet. Activating tensions are strongly influenced by amphipathic substances, and therefore the channel can be used as an endogenous sensor of drug partitioning into the native bacterial membrane. In this project we combine experimental and computational efforts of three groups aimed to explore different sensing modalities of MscS and approach the practical design of a lateral pressure sensor. More specifically, we propose to (1) simulate intercalation of several biologically active compounds into the lipid bilayer and compute changes in lateral pressure profiles using Molecular Dynamics. We will then use these results to simulate MscS expansion to identify intermolecular interactions in the channel that can influence sensitivity to asymmetric tension. (2) Based on these results, we will re-engineer MscS for higher sensitivity and stability. The channel will be calibrated in the presence of substances causing known pressure shifts determined using independent surface chemistry techniques, and then used for practical screening and characterization of several antibiotics and their synthetic analogs. In order to understand the mechanism of MscS inactivation by cytoplasmic crowding, we will (3) computationally explore the conformational dynamics of the hollow cage domain, excluded volumes and compressibilities in different conformations, and the coupling with the pore-lining helices. (4) Conformations identified by computations as functionally important will be tested experimentally through mutagenesis and detailed patch-clamp analysis in the presence of crowding agents. The project will establish the very first sensor-based platform for monitoring incorporation and permeation of amphipathic substances through native bacterial membranes. It will also reveal the allosteric interplay between the membrane-embedded and cytoplasmic domains of MscS, and the biophysical principle by which cells measure the extent of cytoplasmic hydration, thus opening the opportunity for the design of bio-inspired osmosensors.